Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Clinical Criteria as Predictive Factors of Response to Primary Hormone Therapy in Locally Advanced Breast Cancer

This study investigates the efficacy of clinical criteria in selecting patients for primary tamoxifen therapy. A total of 60 breast cancer patients with large primary tumors and unknown hormonal receptor status were subjected to primary hormone therapy. Inclusion criteria were age over 60 years old or menopausal status for at least 10 years and no clinical evidence of inflammatory disease and fast tumor growth. The objective response rate was 55%. There was a positive correlation between the lack of clinical response and axillary lymph node metastasis (p = 0.009). Patients with objective response had significantly improved disease-free (p = 0.045) and overall (p = 0.0002) survival over those who did not have response to hormonal therapy. In multivariate analysis, the clinical response to therapy was the most powerful prognostic factor. This analysis demonstrates that clinical criteria were very effective predictor of response to neo-adjuvant hormone therapy in large breast tumors for postmenopausal women. Response to therapy is the major prognostic factor in primary tamoxifen-treated breast cancer.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Tumor necrosis factor region polymorphisms are associated with AIDS and with cytomegalovirus retinitis

Background: The tumor necrosis factor (TNF) gene is located within the highly polymorphic major histocompatibility complex region, exhibiting the -308 CA promoter region polymorphism and six microsatellites (TNFa-f) spanning the region nearby the TNF locus. Objective: In the present Study, we evaluated the frequency of -308 CA and TNFa-e polymorphisms and respective haplotypes (in chromosomal sequence: TNFd-TNFe-308GA-TNFc-TNFa-TNFb), in 222 patients with AIDS, 52 of whom exhibited cytomegalovirus retinitis, and in 202 healthy HIV-negative individuals. Method: TNF microsatellite and single nucleotide polymorphism typings were performed by PCR followed by polyacrylamide gel electrophoresis. Results: The TNF-308A allele and the 4-3-C-2-7-1 haplotype were associated with susceptibility to AIDS, whereas the TNFb4 allele and the 3-3-C-1-11-4 haplotype were associated with protection against AIDS development. The TNFc2 allele and the 4-1-G-2-2-1 haplotype, which contains the TNFc2 allele, were associated with cytomegalovirus retinitis. Conclusion: This study highlights that polymorphic sites spanning the region nearby the TNF locus are associated with AIDS per se and with cytomegalovirus retinitis in AIDS patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGAS gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus-transformed B-cell lines - A potential therapy for posttransplant lymphoproliferative disease

Background. Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. Method. This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABRA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. Results. In vitro treatment of lymphoblastoid cell lines with ABRA showed that they were effectively killed by low doses of the drug (ID50 2-5 mug/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitt's lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. Conclusion. These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells

Background: The response of hepatocellular carcinoma (HCC) to therapy is often disappointing and new modalities of treatment are clearly needed. Active immunotherapy based on the injection of autologous dendritic cells (DC) co-cultured ex vivo with tumor antigens has been used in pilot studies in various malignancies such as melanoma and lymphoma with encouraging results. Methods: In the present paper, the preparation and exposure of patient DC to autologous HCC antigens and re-injection in an attempt to elicit antitumor immune responses are described. Results: Therapy was given to two patients, one with hepatitis C and one with hepatitis B, who had large, multiple HCC and for whom no other therapy was available. No significant side-effects were observed. The clinical course was unchanged in one patient, who died a few months later. The other patient, whose initial prognosis was considered poor, is still alive and well more than 3 years later with evidence of slowing of tumor growth based on organ imaging. Conclusions: It is concluded that HCC may be a malignancy worthy of DC trials and sufficient details in the present paper are given for the protocol to be copied or modified. (C) 2002 Blackwell Publishing Asia Pty Ltd.

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Inhibition of early tumor growth requires Jα18-positive (natural killer T) cells

The role of natural killer T (NKT) cells in the immune response to tumor cells has been largely unexplored. As a model of adoptive tumor immunotherapy, cells from the draining lymph nodes of mice immunized with a tumor-specific or irrelevant antigen were transferred to naive recipients with established tumor. Inhibition of early tumor growth (day 4) required the transfer of both CD8(+) and Jalpha18(+) (NKT) cells from immunized animals without regard to immunogen. In contrast, CD8(+) cells, but not Jalpha18(+) cells, were necessary for the inhibition of late tumor growth (day 8). Thus, the developing tumor changes in sensitivity to NKT-mediated events and the role for NKT cells cannot be replaced by the presence of tumor-specific cells during early tumor growth. This suggests that recruitment/activation of Jalpha18(+) NKT cells is an important consideration during the immune therapy of early stage tumors.

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

Biochemistry of adipose tissue: an endocrine organ

Adipose tissue is no longer considered to be an inert tissue that stores fat. This tissue is capable of expanding to accommodate increased lipids through hypertrophy of existing adipocytes and by initiating differentiation of pre-adipocytes. Adipose tissue metabolism exerts an impact on whole-body metabolism. As an endocrine organ, adipose tissue is responsible for the synthesis and secretion of several hormones. These are active in a range of processes, such as control of nutritional intake (leptin, angiotensin), control of sensitivity to insulin and inflammatory process mediators (tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), resistin, visfatin, adiponectin, among others) and pathways (plasminogen activator inhibitor 1 (PAI-1) and acylation stimulating protein (ASP) for example). This paper reviews some of the biochemical and metabolic aspects of adipose tissue and its relationship to inflammatory disease and insulin resistance.

Presence of Circulating Levels of Interferon-g, Interleukin-10 and Tumor Necrosis Factor-a in Patients with Visceral Leishmaniasis

Experimental murine L. major infection is characterized by the expansion of distinct CD4+ T cell subsets. The Th1 response is related to production of IFN-g and resolution of infection, whereas Th-2 response with production of IL-4 and IL-10 and dissemination of infection. The objective of this study was to measure the circulating levels of IFN-g, IL-10 and TNF-a in patients with visceral leishmaniasis (VL) before, during and at the end of therapy and to examine the association between cytokine levels and activity of VL. Fifteen patients with VL were evaluated. The cytokine determinations were done by using the enzyme-linked immunoassay (ELISA) before, during and at the end of therapy. At baseline, we detected circulating levels of IFN-g in 13 of 15 patients (median = 60 pg/ml); IL-10 in 14 of 15 patients (median = 141.4 pg/ml); and TNF-a in 13 of 14 patients (median = 38.9 pg/ml). As patients improved, following antimonial therapy, circulating levels of IL-10 showed an exponential decay (y = 82.34 e–0,10367x, r = –0.659; p < 0.001). IFN-g was no longer detected after 7/14 days of therapy. On the other hand, circulating levels of TNF-a had a less pronounced decay with time on therapy, remaining detectable in most patients during the first seven days of therapy (y = 36.99-0.933x, r = –0.31; p = 0.05). Part of the expression of a successful response to therapy may, therefore, include reduction in secretion of inflammatory as well as suppressive cytokines. Since IL-10 and IFN-g are both detected prior to therapy, the recognized cellular immune depression seen in these patients may be due to biological predominance of IL-10 (type 2 cytokine), rather than lack of IFN-g (type 1 cytokine) production.

Evaluation of Bone Remodeling in Hemodialysis Patients: Serum Biochemistry, Circulating Cytokines and Bone Histomorphometry

BACKGROUND: To optimize the noninvasive evaluation of bone remodeling, we evaluated, besides routine serum markers, serum levels of several cytokines involved in bone turnover. METHODS: A transiliac bone biopsy was performed in 47 hemodialysis patients. Serum levels of intact parathyroid hormone (iPTH; 1-84), total alkaline phosphatases (tAP), calcium, phosphate and aluminum (Al) were measured. Circulating levels of interleukin-6 (IL-6), IL-1 receptor antagonist (IL-1Ra) and soluble IL-6 receptor (sIL-6r) were determined using ELISA. Circulating IL-1beta, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor-alpha (TNF-alpha) were simultaneously quantified by flow cytometric immunoassay. RESULTS: Patients with low/normal bone formation rate (L/N-BFR) had significantly lower serum iPTH (p<0.001) and tAP (p<0.008) and significantly higher Al (p<0.025) than patients with high BFR. Serum calcium and phosphorus, however, did not differ (p=NS). An iPTH >300 pg/mL in association with tAP >120 U/L showed low sensitivity (58.8%) and low negative predictive value (44.0%) for the diagnosis of high BFR disease. An iPTH <300 pg/mL in association with normal or low tAP, <120 U/L, was associated with low sensitivity (66.7%) but high specificity (97.1%) for the diagnosis of L/N-BFR. Serum IL-1, IL-6, IL-12p70 and TNF-alpha were positively correlated with BFR, serum IL1-Ra and IL-10 with bone area, and by multiple regression analysis, tAP and IL-6 were independently predictive of BFR. CONCLUSIONS: Significant associations were found between several circulating cytokines and bone histomorphometry in dialysis patients. The usefulness of these determinations in the noninvasive evaluation of bone remodeling needs to be confirmed in larger dialysis populations.